Use of terpenoids in the treatment or prevention of fibrotic diseases

ABSTRACT

The present invention relates to a method of preventing or treating a fibrotic condition, comprising administering an effective amount of composition to a subject in need thereof; wherein the composition comprises triterpenes extracted from  Antrodia camphorate  or  Anisomeles indica.

FIELD OF THE INVENTION

The invention relates to herbal terpenoids from Antrodia camphorata andAnisomeles indica extract, particularly to a medicinal and edibleformula to moderate fibrotic diseases effectively.

BACKGROUND OF THE INVENTION

Fibroproliferative disorders are troubling problems for an increasingnumber of individuals and is a common pathological sequela of manypersistent inflammatory diseases, such as pulmonary fibrosis,progressive kidney disease, liver cirrhosis, atherosclerosis and benignprostatic hyperplasia.

Impaired renal repair after acute kidney injury induces fibrosis whichmay ultimately lead to the development of chronic kidney disease. Kidneyinjury activates multipotent progenitor cells to repair tissue. However,these cells become dysfunctional and induce fibrogenic repair as theinjury sustains initiating kidney fibrosis. The pathogenesis of renalfibrosis is a progressive process that ultimately leads to end-stagerenal failure, a devastating disorder that requires dialysis or kidneytransplantation.

Non-alcoholic fatty liver disease (NAFLD) is a leading form of chronicliver disease with large unmet need. Non-alcoholic steatohepatitis(NASH), a progressive variant of NAFLD, can lead to fibrosis, cirrhosis,and hepatocellular carcinoma. NAFLD and NASH are entities that arebecoming subject of interest of the medical community in general,especially because of the increased prevalence of diabetes and obesityin the world population. Clinical evaluation of every patient withabnormal aminotransferase levels should take into account non-alcoholicfatty liver and its spectrum, especially if the subject is obese ordiabetic. The prognosis of simple NAFLD is generally benign, but ifthere is fibrosis, ballooning of the hepatocytes, inflammation andMallory bodies there is risk to progression to cirrhosis.

Autoimmune hepatitis (AIH) is a chronic liver disease without a clearetiology but can be characterized by hepatocellular inflammation. SevereAIH may progress into liver cirrhosis, hepatocellular carcinoma, or evendeath. Cirrhosis develops in as many as 40% of treated patients withautoimmune hepatitis depending on the length of observation.Anti-fibrotic therapies are emerging that can supplement theanti-inflammatory and immunosuppressive actions of current regimens, andthese regimens promise to re-direct the objectives of treatment inautoimmune hepatitis to the prevention, stabilisation and reversal ofhepatic fibrosis.

Atherosclerosis, which is one of the primary causes of the developmentof cardiovascular disease, is associated with vascular fibrosis.Vascular fibrosis involves accumulation of extracellular matrix (ECM)proteins, particularly collagen and fibronectin in the vascular mediaand contributes to structural remodeling and scar formation. A lack ofelastin or excessive collagen in the vascular wall leads to vascularfibrosis and increased stiffness.

In benign prostatic hyperplasia, the deposition of collagen fibers inprostate is for replacing broken myofibers, however results in stiffnessand weakness of the muscular tissue and deposition of prostatic fluid ingland tubes. Prostatic fibrosis plays a central role in the developmentof bladder outlet obstruction in aging men.

Medicinal fungus Antrodia camphorata (AC) is a well-known Chinese folkmedicine, known to possess numerous biological activities, especially ananti-tumor effect in in vitro cancer cells and in vivo animal models. Itis considered an efficient alternative phyto-therapeutic agent or anadjuvant to cancer treatment and immune-related diseases given itsdiverse bioactive compounds. To date, a total of 225 compounds have beenisolated, identified, and structurally elucidated, includingmacromolecules (nucleic acids, proteins, and polysaccharides), smallmolecules (benzenoids, lignans, benzoquinones, and maleic/succinic acidderivatives), terpenoids (lanostane triterpenes, ergostane triterpenes,diterpenes, monoterpenes, and steroids), nucleotides (nucleobase andnucleoside), fatty acids, and fatty acid esters.

Cumulative in vitro and in vivo studies have revealed its anti-diabeticand anti-hyperlipidemic, anti-hypertensive, anti-inflammatory,antioxidant, antimicrobial, cardiovascular disease preventive,immunomodulatory, hepatoprotective, and neuroprotective effects.However, the efficacy of Antrodia camphorata and its components in thetreatment of fibrosis has not been evaluated.

Anisomeles indica commonly known as ‘Indian Catmint’ is a source ofmedicinally active compounds and have various pharmacological effects.The plant is used traditionally as an analgesic, anti-inflammatory andin skin problems. Medicinally it has been proven to possess variouspharmacological activities like antioxidant, antimicrobial, anti-HIV,anti-Helicobacter pylori and anti-cancer activity. It is also used inchronic rheumatism. Further studies reveal the presence of variousphytochemical constituents mainly triterpenes, β-sitosterol,stigmasterol, flavones, apigenin and ovatodiolides etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the separation of antcin K, dehydrosulphurenicacid/sulphurenic acid, versisponic acid D and dehydroeburicoic acid fromAntrodia camphorata extract

FIG. 2 Protective effects of Antrodia camphorata Extract and Compoundson cisplatin-induced kidney damage in AKI mice. To analyze the effectsof Antrodia camphorata Extract and compounds, mice are given daily for 7days, starting from 3 weeks after first dose of cisplatin, andsacrificed at 4 weeks. The morphological changes in the kidneys (A).Blood urea nitrogen (BUN) levels (B). Serum creatinine (CRE) levels (C).The data are presented as the means±S.E.M (n=5). ###denotes p<0.001compared with sample of control group. **p<0.01 and ***p<0.001 comparedwith cisplatin group.

FIG. 3 Protective effects of Antrodia camphorata Extract and Compoundson cisplatin-induced kidney damage in AKI mice. To analyze the effectsof Antrodia camphorata Extract and compounds, mice are given daily for 7days, starting from 3 weeks after first dose of cisplatin, andsacrificed at 4 weeks. Kidneys stained with H&E. After cisplatinchallenge, kidneys in each group are prepared for histologicalevaluation. Representative histological section of the kidneys isstained by H&E staining, magnification (400×). The data are presented asthe means±S.E.M (n=5). ###denotes p<0.001 compared with sample ofcontrol group. **p<0.01 and ***p<0.001 compared with cisplatin group.Tubular cell necrosis is marked with arrows; the bar indicates 50 μm.

FIG. 4 Antrodia camphorata Extract and Compounds regulated (A) TNF-α,(B) IL-1β, (C) IL-6 (D) TGF-β and (E) albumin in serum. Serum levels ofTNF-α, IL-1β, IL-6, TGF-β and albumin are determined by commercial ELISAkits. Data are represented as mean±S.E.M. (n=5). ###denotes p<0.001compared with sample of control group. **p<0.01 and ***p<0.001 comparedwith cisplatin-only group.

FIG. 5 Effects of ARH005-EA (A) and ARH (B) on cisplatin-induced TWEAK,α-SMA, P53 and P21 signaling expression in kidneys. Protein levels ofTWEAK, α-SMA, P53 and P21 protein expression in kidney homogenates areevaluated by western blot analysis after cisplatin challenge.

FIG. 6 depicts the process of CCl₄-induced fibrosis model

FIG. 7 depict (A) delta weight, (B) liver weight, and (C)liver/body-weight ratio, respectively.

FIG. 8 depict serum levels of (A) AST, (B) ALT, and (C) AST/ALTrespectively in rats following CCl₄-induced hepatic damage.

FIG. 9 depict (A) inflammation, (B) vacuolation, (C) necrosis, (D)fibrosis, (E) total histological score in liver.

FIG. 10 Representative histological section of the liver is stained byH&E staining.

FIG. 11 depicts the process of Con A (concanavalin A) induced acutehepatitis model.

FIG. 12 depict the process of Ovatodiolide (AR100-DS1) on GOT, GPT andbody weight. (A) Serum GOT and (B) serum GPT 24 h after 15 mg/kg Con Achallenge. (C) body weight before and after 15 mg/kg Con A challenge.Data are presented as mean f SEM (n=9). *p<0.05 versus Veh by the ttest. Veh, vehicle; Dex, dexamethasone.

FIG. 13 Effects of Ovatodiolide (AR100-DS1) on liver injury. Liverhistopathology of (A) Naïve, (B) 15 mg/kg Con A (Veh), (C) 2019-0321-1and (D) dexamethasone, and (E) histopathological scores of necrosis.Data are presented as mean f SEM (n=9). ***p<0.001 versus Veh by theStudent's t test. Veh, vehicle; Dex, dexamethasone.

FIG. 14 depicts the process of atherosclerosis rabbit model.

FIG. 15 depicts initial and final average body weight of rabbits. †and * indicate a P<0.05 as compared with the control group and HF group,respectively.

FIG. 16 depict changes in AST, ALT, BUN between WO groups in each groupof rabbits † and * indicate a P<0.05 as compared with the control groupand HF group, respectively.

FIG. 17 depict changes in TG, TC, HDL-C, LDL-C between WO groups in eachgroup of rabbits † and * indicate a P<0.05 as compared with the controlgroup and HF group, respectively.

FIG. 18 depict changes in AST, ALT, BUN between W4 groups in each groupof rabbits † and * indicate a P<0.05 as compared with the control groupand HF group, respectively.

FIG. 19 depict changes in TG, TC, HDL-C, LDL-C between W4 groups in eachgroup of rabbits. † and * indicate a P<0.05 as compared with the controlgroup and HF group, respectively.

FIG. 20 depict changes in AST, ALT, BUN between W8 groups in each groupof rabbits † and * indicate a P<0.05 as compared with the control groupand HF group, respectively.

FIG. 21 depict changes in TG, TC, HDL-C, LDL-C between W8 groups in eachgroup of rabbits. † and * indicate a P<0.05 as compared with the controlgroup and HF group, respectively.

FIG. 22 depict changes in AST, ALT, BUN between W12 groups in each groupof rabbits † and * indicate a P<0.05 as compared with the control groupand HF group, respectively.

FIG. 23 depict changes in TG, TC, HDL-C, LDL-C between W12 groups ineach group of rabbits. † and * indicate a P<0.05 as compared with thecontrol group and HF group, respectively.

FIG. 24 depict histopathochemical examination of aortic fatty streaklesions in the hypercholesterolemic rabbit model after the 12-weekstudy.

FIG. 25 depicts HE staining of coronary artery sections after sacrificein each group of rabbits.

FIG. 26 depicts HE staining of coronary arteries after sacrifice in eachgroup of rabbits. N, neointima layer; M, media layer.

FIG. 27 depicts the manifestation of vascular restenosis is presented asthe ratio of neointima-to-media area (N/M ratio) N, neointima layer; M,media layer. *p<0.05, **p<0.01, and ***p<0.001 compared with HFD group,respectively.

FIG. 28 depicts histopathochemical examination of heart tissues in thehypercholesterolemic rabbit model after the 12-week study.

FIG. 29 depicts photographs of liver appearance in thehypercholesterolemic rabbit model after the 12-week study.

FIG. 30 depicts histopathochemical examination of liver tissues in thehypercholesterolemic rabbit model after the 12-week study.

FIG. 31 depicts the animal's body weight and lung weight.

FIG. 32 depicts histopathological changes of the lungs inbleomycin-induced lung fibrosis in mice.

FIG. 33 depicts Masson's Trichrome staining of the lung inbleomycin-induced lung fibrosis in mice.

FIG. 34 depicts the effects of A. camphorata Extract and Compounds onhydroxyproline content in pulmonary damage of mice induced by bleomycin.

FIG. 35 depict A. camphorata Extract and compounds regulated (A) TNF-α,(B) IL-1β, (C) IL-6 (D) and TGF-β(E) in BALF.

FIG. 36 depicts the effects of A. camphorata Extract and compoundsregulated on lung MPO activity in BLM-induced mice.

DETAILED DESCRIPTION OF THE INVENTION

For the convenience of the description of the present invention, thecentral idea expressed in the above summary of the invention isexpressed by way of specific examples. Various items in the embodimentsare depicted in terms of ratios, dimensions, amounts of deformation, ordisplacements that are suitable for illustration, and are not drawn tothe proportions of actual elements, as set forth above.

The term “terpenes” refers to a large and diverse class of organiccompounds, whose basic structure follows a general principle:2-Methylbutane residues, less precisely but usually also referred to asisoprene units, (C₅)_(n), build up the carbon skeleton of terpenes.About 30 000 terpenes are known at present in the literature. Dependingon the number of 2-methylbutane (isoprene) subunits one differentiatesbetween hemi- (C₅), mono- (C₁₀), sesqui- (C₁₅), di- (C₂₀), sester-(C₂₅), tri- (C₃₀), and tetraterpenes (C₄₀).

The terms “subject,” “individual.” “host, and “patient” are usedinterchangeably herein to refer to a living animal, including a humanand a non-human animal. The Subject may, for example, be an organismpossessing immune cells capable of responding to antigenic stimulation,and stimulatory and inhibitory signal transduction through cell Surfacereceptor binding. The Subject may be a mammal. Such as a human ornon-human mammal, for example, dogs, cats, pigs, cows, sheep, goats,horses, rats, and mice. The term ‘subject does not preclude individualsthat are entirely normal with respect to a disease, or normal in allrespects.

The term “treatment” refers to a therapeutic or preventative measure.The treatment may be administered to a subject having a medical disorderor who ultimately may acquire the disorder, in order to prevent, cure,delay, reduce the severity of or ameliorate one or more symptoms of adisorder or recurring disorder, or in order to prolong the Survival of asubject beyond that expected in the absence of such treatment.

The term “therapeutically effective amount’ means the amount of thesubject compound that may elicit a desired response, for example, abiological or medical response of a tissue, System, animal, or humanthat is sought, for example, by a researcher, veterinarian, medicaldoctor, or other clinician.

Determination of biochemical parameters Serum creatinine and serum ureaare assessed using colorimetric kits according to manufacturer'sinstructions. Kits of the former markers are purchased from (HUMANDiagnostics Worldwide, Magdeburg, Germany) with a chemical analyzer(Roche Diagnostics, Cobas Mira Plus, Rotkreuz, Switzerland).

Kidney histopathology The anterior portion of the left lateral liverlobe from each mouse is fixed in 10% formaldehyde phosphate buffer,embedded in paraffin, cut into 5 μm sections, and then treated with anhematoxylin and eosin (H&E) stain for histological examination under thelight microscopy (Nikon, ECLIPSE, TS100, Tokyo, Japan). Images arecaptured with a digital camera (NIS-Elements D 2.30, SP4, Build 387) atan original magnification of 400×.

TNF-α, IL-6, and IL-1β cytokines in serum The serum concentration of thepro-inflammatory cytokines (i.e. tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6), and IL-1β) in serum are assessed with relevantenzyme-linked immunosorbent assay (ELISA) kits (Biosource InternationalInc., Sunnyvale, Calif., USA), based on the manufacturer's instructions.

Western blot analysis of the kidney tissues Lysis buffer, composed of0.6% NP-40, 150 mM NaCl, 10 mM HEPES (pH 7.9), 1 mM EDTA, and 0.5 mMPMSF, is used in the homogenization of liver tissues at 4° C. Thehomogenized samples are then centrifuged at 3000 revolutions per minute(rpm) at 4° C. for 10 min to obtain the supernatant. The equal totalcellular protein amounts of supernatant are determined by the proteinstandard of bovine serum albumin (BSA). Protein samples (50 μg) areresolved by denaturing 10% sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) using standard methods, and then aretransferred onto PVDF membranes (Immobilon, Millipore, Bedford, Mass.,USA) for electroblotting and blocking with 10% skim milk. The membranesare incubated with an appropriate dilution of specific primaryantibodies at 4° C., washed three times with TBS/tween (TBST) buffer,and subsequently incubated for 1 h at 37° C. with horseradishperoxidase-conjugated secondary antibodies (overnight). The membranesare washed three times before examination for immuno-reactive proteinsby enhanced chemiluminescence (ECL) reagent (Thermo Scientific, Hudson,N.H., USA). Band intensity on scanned films are quantified andrepresented as relative intensity by comparing with the control groupusing Image J Software (NIH, Bethesda, Md., USA).

Statistical analysis Data obtained from animal experiments are expressedas the means and standard errors of the means (±S.E.M.). Student'st-test are used to examine the differences among multiple groups orbetween two groups. Statistical significance is expressed as *p<0.05,**p<0.01 and ***p<0.001.

Example 1. Preparation of Antrodia camphorata Extract

100 grams of Antrodia camphorata fruiting body is reflux with methanolfor 6 hours, and the extract is collected and dried, thereby obtaining atotal of 15 grams of methanol extract of Antrodia camphorata.

Example 2. Preparation of Active Ingredients: Antcin K,Dehydrosulphurenic acid/sulphurenic acid, Versisponic acid D andDehydroeburicoic acid

The methanol extract of Antrodia camphorata is further separated bysilica column chromatography using n-hexane/ethyl acetate/methanol aseluent to provide the fractions (shown in FIG. 1 ):

ARH101-DS1 (RS-Antcin K),

ARH101-DS2 (Dehydrosulphurenic acid/Sulphurenic acid),ARH101-DS3 (Versisponic acid D) and

ARH101-DS4 (Dehydroeburicoic Acid).

Example 3. Preparation of AR003 Extract

100 grams of Antrodia camphorata (petri dish culture) is reflux withmethanol for 6 hours, and the extract is collected and dried underreduced pressure to obtain 15 grams of the Antrodia camphorate ARH003extract.

Example 4. Preparation of AR003-E Extract

200 grams of Antrodia camphorata (petri dish culture) is reflux withethanol for 6 hours, and the extract is collected and dried, therebyobtaining a total of 18 grams of ethanol extract of AR003-E Antrodiacamphorata.

Example 5. Preparation of AR004 Extract

100 grams of Antrodia camphorata (wood culture) is reflux with methanolfor 6 hours, and the extract is collected and dried under reducedpressure to obtain the Antrodia camphorate ARH004 extract.

Example 6. Preparation of AR005-EA Extract

100 grams of Antrodia camphorata (solid culture) is reflux with ethylacetate for 6 hours, and the extract is collected and dried, therebyobtaining a total of 12 grams of EA extract of Antrodia camphorata.

Example 7. Preparation of Anisomeles indica Extract

The Anisomeles indica extract is prepared by the following process: (1)An ethanol extract of Anisomeles indica is taken, added into asilica-filled chromatographic column, and subjected to a gradientelution with the eluents “n-hexane/ethyl acetate”, “hexane/ethylacetate/methanol” and “methanol” to obtain an Anisomeles indica partingliquid. (2) The Anisomeles indica parting liquid is separated by usingsilica-filled chromatographic column, and subjected to a gradientelution with the eluents “dichloromethane”, “dichloromethane/methanol”and “methanol” to obtain a separated concentrating substance; (3) theseparated concentrating substance is recrystallized with the solvent“n-hexane/ethyl acetate” to obtain the Anisomeles indica crystallite.

Example 8. Preparation of Active Ingredients: Ovatodiolide (AR100-DS1)

200 g of ethanol extract of Anisomeles indica is taken, added into asilica-filled chromatographic column (10×15 cm), and subjected to agradient elution with 1200 ml of each of the eluents: “n-hexane/ethylacetate (with a ratio of 10/1, 5/1, 3/1, 1/1)”, “hexane/ethylacetate/methanol (with a ratio of 6/4/1, 3/2/1)” and “methanol” toobtain 140 g of an initial parting liquid.

The 140 g of the initial parting liquid is separated by usingsilica-filled chromatographic column (10×15 cm), and subjected to agradient elution with 1000 ml of each of the eluents: “dichloromethane”,“dichloromethane/methanol (with a ratio of 10/1, 5/1, 7/3)” and“methanol” to obtain a separated concentrating substance. The separatedconcentrating substance is further recrystallized with the solvent:“n-hexane/ethyl acetate” to obtain a crystal. The crystal is identifiedas a diterpenoid compound whose chemical structure is ovatodiolide by anuclear magnetic resonance spectroscopy (H1-NMR). The crystal iscompared with the standard product of ovatodiolide by high performanceliquid chromatography (HPLC) analysis, and is recognized to haveovatodiolide compounds.

Metabolites from Ovatodiolide (AR100-DS1):

+O, +Cysteine: m/z:466, M2, M3, M4 +Glutathione: m/z:636, M6, M7 +O:m/z:345, M8, M9

Com- pound No. Compound name and structure AR101- DS1

AR101- DS2

AR101- DS3

AR101- DS4

AR100- DS1

AR100- DS4

AR100- DS5

AR100- DS6

AR100- DS7

AR100- DS8

AR100- DS9

AR100- DS10

AR100- DS11

AR100- DS12

AR100- D13

Example 9. Mice Model of Cisplatin-Induced Renal Injury

Seven-to-eight-week-old male C57BL/6 mice are obtained from the BioLASCOTaiwan Co., Ltd. (Taipei, Taiwan). The animals are housed in Plexiglascages at a constant temperature of 22±1° C. and a relative humidity of55±5% on a 12 h dark-light cycle for at least 2 weeks before theexperiment. Animals are provided food and water ad libitum. Allexperimental procedures are performed according to the guidelines of theInstitutional Animal Ethics Committee, and the protocol is approved bythe Committee for the Purpose of Control and Supervision of Experimentson Animals.

Renal fibrosis is induced via multiple injections of low-dose cisplatin.Intraperitoneal injections of cisplatin (5 mg/kg/injection; P4394,Sigma-Aldrich, St Louis, Mo.) are performed at 0, 1, and 3 weeks, for atotal of 3 injections. Mice are sacrificed at 6 weeks after the firstdose of cisplatin (n=6). To analyze the effects of samples, mice aregiven daily intraperitoneal injections for 7 days, starting from 4 weeksafter first dose of cisplatin, and sacrificed at 4 weeks (n=6).

Example 10. Antrodia camphorata Extract and Compounds Reduced RenalDysfunction and Histopathological Changes in Cisplatin-Induced Mice

The morphological changes in the kidneys are shown in the FIG. 2A. CREand BUN are hallmarks of kidney function. FIGS. 2B and 2C show that,compared to the control group, cisplatin injection at the three 10 mg/kgCP doses (at 0, 1, 3 weeks) highly increased the serum CRE and BUNlevels (p<0.001), indicating the generation of nephrotoxicity in thecisplatin-treated mice. Treatment with ARH005-EA and ARH003-E at dosesof 1000 mg/kg and compounds (AR101-DS4 and AR100-DS1) exerted asignificant renal protection effect in a dose-dependent manner, asdemonstrated by the normalization of CRE and BUN (p<0.001) compared tocisplatin-stimulated group.

Example 11. Antrodia camphorata Extract and Compounds Alleviates RenalDysfunction and Renal Injury Induced by Multiple Cisplatin Treatment

The histopathological changes are analyzed to determine whether Antrodiacamphorata extract and compounds affected renal failure incisplatin-stimulated mice. The kidney tissue of the control group iscompletely normal and characterized by a transparent tubular andglomerular structure with clear and normal nuclei. Kidneys had severekidney damage in the cisplatin-stimulated mice, inducing tubularepithelial damage, inflammatory cell infiltration, tubular cellswelling, formation of intratubular casts, and tubular dilatation.However, treatment with Antrodia camphorata extract (AR005-EA) at dosesof 1000 mg/kg and compounds (AR100-DS1) significantly improved necrosisand inflammatory infiltrating cells in the kidney tissue (see FIG. 3 ).

Example 12. Antrodia camphorata Extract and Compounds Alerted theCisplatin-Induced Changes in Pro-Inflammatory Cytokines and Albumin

Evaluation of proinflammatory cytokine TNF-α, IL-1β, IL-6 and TGF-βlevels in serum are performed by ELISA. Cisplatin-treated kidney injurymice had significantly increased NO, TNF-α, IL-1β, and IL-6 levels inserum, compared to the control group (FIGS. 4A-4E, respectively).Treatment with Antrodia camphorata Extract (AR005-EA) at doses of 1000mg/kg and compounds (AR100-DS1) significantly improved necrosis andinflammatory infiltrating cells in the kidney tissue treatment improvedNO, TNF-α, IL-1β, and IL-6 production after cisplatin challenge.

Example 13. Inhibition of Cisplatin-Induced Renal Injury TWEAK, α-SMA,P53 and P21 Protein Expression

The results are examined whether pretreatment with Antrodia camphorataExtract (ARH005-EA) and compounds (AR100-DS1) inhibitedcisplatin-induced TWEAK, α-SMA, P53 and P21 protein expression. Theresults revealed that treatment with ARH005-EA and ARH inhibited theprotein expression of TWEAK, α-SMA, P53 and P21 in the kidney tissuesafter the cisplatin challenge (FIGS. 5A and 5B).

Example 14. CCl₄-Induced Chronic Liver Fibrosis in Rat

As shown in FIG. 6 , eight-week-old male SD rats are administered CCl₄at 0.4 mg/kg as twice per week for 8 weeks. Blood samples are collectedat week 0, 2, 4, 6, and 8. The animals are sacrificed at the end of the8-week for histopathological examinations. FIGS. 7A, 7B, 7C depict deltaweight, liver weight, and liver/body-weight ratio, respectively. Theliver weight of Naïve group is not significantly different from that ofVehicle group; however, the liver/body-weight ratio of Naïve group issignificantly smaller than that of Vehicle group. The liver weight andliver/body-weight ratio of 50 mg/kg AR100-DS1 group are significantlylarger comparing to that of Vehicle and Naïve group.

Example 15. Serum Liver Enzymes Profile

The level of clinical biochemistry such as aspartate aminotransferase(AST), alanine aminotransferase (ALT), is evaluated to determine theenzymatic activities of the livers of the control groups and theexperimental groups (shown in FIGS. 8A-8C). The level of AST, ALT andAST/ALT ratio of Naïve group showed no significant changes during theexperiment. The serum AST and ALT level of the animals in each testgroup increased significantly with the progress of the experiment;however, compared with the vehicle group, less AST and ALT increases canbe observed in the 50 mg/kg AR100-DS1 group at W6 and W8.

Example 16. Liver Histological Evaluation

After 8 weeks of CCl₄ induction, the Vehicle group significantlysuffered from liver injury such as increased AST and ALT, decreasedAST/ALT ratio, inflammation, fibrosis, vacuolation and necrosis. Asshown in FIGS. 9A-9E and 10 , the liver of 50 mg/kg AR100-DS1 group hasa smooth surface without atrophy and sclerosis, and the liver weight andliver/body weight-ratio are significantly larger than that of Vehicleand Naïve group. Overall, AR100-DS1 has been shown to have the potentialto partially repair CCl₄-induced liver injury.

Example 17. Effects of Ovatodiolide (AR100-DS1) on Con A (ConcanavalinA) Induced Acute Hepatitis in BALB/c Mice

Intravenous injection of concanavalin A (Con A) is a widely usedstrategy to study T cell mediated hepatitis. Con A is a lectin that canactivate CD4⁺ T cells, produce cytokines, and lead to liver cell damage.Dexamethasone (Dex) is a long acting synthetic corticosteroid, and hasbeen used as anti-inflammatory and immunosuppressive medication. Theeffects of Ovatodiolide (AR100-DS1) on serum glutamic-pyruvictransaminase (GOT), glutamic-oxaloacetic transaminase (GPT), circulatingcytokines and liver histopathology on Con A-induced acute hepatitis areevaluated in BALB/c mice.

Con A and Dex were purchased from Sigma Aldrich (USA). ProcartaPlex™immunoassays kit was purchased from Corning Inc. (USA). GOP and GPT FujiDri-Chem slides were purchased from Winning Medical Inc. (Taiwan).

Male BALB/c mice (7-9 weeks old) were purchased from BioLASCO TaiwanCo., Ltd or National Laboratory Animal Center (NLAC, Taiwan). Animalsare housed five per cage with food and water provided ad libitumthroughout the experiments. Room temperature is maintained at 23±2° C.with an alternating 12 h light dark cycle. Animals are acclimatized forone week to minimize the effect of stress before the experiments. Allexperimental protocols involving animals and their care are approved bythe Institutional Animal Care and Use Committee (IACUC) in ITRI(ITRI-IACUC-2018-041 and ITRI-IACUC-2018-050; accredited by AAALAC) andare carried out according to the regulations of the Council ofAgriculture, Taiwan.

Con A is dissolved in pyrogen free saline at a concentration of 3 mg/mLand intravenously injected at a dose of 15 mg/kg or 20 mg/kg of bodyweight to induce hepatitis. Ovatodiolide (AR100-DS1) and Dex are orallyadministered 30 min before and then 4 h and 8 h after Con A treatment.Blood and liver tissues are collected 24 h after Con A treatment (FIG.11 ). Serum are stored at 80° C. until analysis.

To assess the level of hepatocellular injury after Con A treatment,serum GPT and GOT levels are measured by Fuji Dri-Chem slides (Fuji,Japan). The serum of the same group is pooled for cytokine assay.Cytokine levels are measured by ProcartaPlex™ immunoassays kit accordingto manufacturer's instructions. Data are presented as mean f SEM. T testis used to analyze the differences between drug and vehicle treatedgroups. The difference is regarded statistically significant when pvalue is less than 0.05. Ovatodiolide (AR100-DS1) at 50 mg/kgsignificantly reduced GPT level that is increased by Con A (109±25 vs368±107 U/L, p<0.05) and slightly improved elevation of GOT (261±45 vs410±56 U/L) (FIG. 12 ).

Liver tissues are fixed in 10% phosphate buffered formaldehyde, embeddedin paraffin, and stained with hematoxylin and eosin (H&E) in order toconfirm tissue lesions. Tissue lesions are examined microscopically by aveterinary pathologist at BioLASCO Taiwan Co., Ltd. The criteria ofseverity grading system for all microscopic lesions are graded from 0 to4 as follows: 0=none; 1=individual cell necrosis; 2=≤30% lobularnecrosis; 3=≤60% lobular necrosis; 4=>60% lobular necrosis. Thehistopathological analysis showed Ovatodiolide (AR100-DS1) amelioratedliver necrosis (score 0.2±0.2 vs 1.4±0.2, p<0.05) (FIG. 13 ). Theresults show that ovatodiolide (AR100-DS1) reduced serum GOP and GPT andattenuated Con A induced liver necrosis.

Example 18. Evaluation of the Efficacy of Antrodia Camphorata Extractand AR101-DS2 in Preventing Atherosclerosis and Liver Fibrosis

Experimental Model

2 to 3 kg male, New Zealand White rabbits are individually caged andhoused in temperature and humidity-controlled rooms. Light-dark cyclesare 12 h each. After several days of acclimation, the animals aresequentially assigned to six feeding groups: standard rabbit chow,standard rabbit chow containing 0.5% cholesterol, standard rabbit chowcontaining both 0.5% cholesterol and 10 mg/kg Lovastatin, standardrabbit chow containing both 0.5% cholesterol and 1% ARH003, standardrabbit chow containing both 0.5% cholesterol and 1% ARH004, standardrabbit chow containing both 0.5% cholesterol and 10 mg/kg AR101-DS2.Except standard rabbit chow, others groups are given standard rabbitchow containing 0.5% cholesterol for 4 weeks (see FIGS. 14-15 ). Thedaily feeding amount for each rabbit is 50 g/kg body weight per day.Diets are administered for 8 weeks, after the animals had adjusted totheir new environment. At the beginning and end of the 12 weeks study,the rabbits are anesthetized by an intramuscular injection of Zoletil 50(1 mL/kg) (Virbac Ltd., France), and blood samples are harvested.Finally, the aortas (from aortic arch to the bifurcation of the iliacarteries) and whole livers are collected from the rabbits after they aresacrificed for further histopathological analyses.

2 to 3 kg male, New Zealand White rabbits (n=30) are divided into thefollowing groups:

(ND) standard rabbit chow, n=5;(HF) standard rabbit chow containing 0.5% cholesterol, n=6;(L) standard rabbit chow containing both 0.5% cholesterol and 10 mg/kgLovastatin, n=4;(AR003) standard rabbit chow containing both 0.5% cholesterol and 1%ARH003, n=5;(AR004) standard rabbit chow containing both 0.5% cholesterol and 1%ARH004, n=5;(AR101-DS2) standard rabbit chow containing both 0.5% cholesterol and 10mg/kg AR101-DS2, n=5;The daily feeding amount for each rabbit is 50 g/kg body weight per day.

Blood Chemistry Analysis

The animals are fasted overnight before blood drawing. The blood iscollected from the marginal ear veins of rabbits into BD Vacutainer EDTABlood Collection Tubes. Plasma is separated by centrifugation at 3,000rpm at 4-C for 10 min. FIGS. 16-23 depict measurements for changes inblood chemistry parameters included serum levels of low-densitylipoprotein (LDL), cholesterol (Chol), triglycerides (TG), glutamateoxaloacetate transaminase (GOT), and glutamate pyruvate transaminase(GPT).

Aortic Fatty Streak Staining

The aortas are opened longitudinally to expose the intimal surface andrinsed gently with normal saline (see FIGS. 24-26 ). Aortas areincubated in 2% (w/v) Sudan IV, rinsed with several concentrations(100%, 90%, 80%, 70%, and 60%) of ethanol for 1 min, and then rinsedwith pure water. The photographs shown in FIG. 28 are acquired using adigital camera (Nikon D80, Japan) and quantified on an Alpha Imager 2200documentation system (Alpha Innotech, USA). The progression of the fattystreak lesions is presented as the percentage of the stained area to thetotal area (FIG. 27 ).

Method

1. Hydrate the cells or tissue:i. Use a microscope slide bearing cryosections or rehydrated tissuesections (see Step 12 in Cutting Sections of Paraffin Embedded Tissues)(Fischer et al. 2008) fixed in either alcohol or an aldehyde-basedfixative.ii. Immerse the slide for 30 sec with agitation by hand in H2O.A rinse in H₂O is important; hematoxylin precipitates with salts andbuffers. The staining can be performed after immunohistochemical orhybridization reactions with nonfluorescent detection systems.2. Dip the slide into a Coplin jar containing Mayer's hematoxylin andagitate for 30 sec.3. Rinse the slide in H₂O for 1 min.Estimate the staining intensity at this point, and repeat Steps 2 and 3if necessary.4. Stain the slide with 1% eosin Y solution for 10-30 sec withagitation.5. Dehydrate the sections with two changes of 95% alcohol and twochanges of 100% alcohol for 30 sec each.Some colorimetric substrates dissolve in alcohol.6. Extract the alcohol with two changes of xylene.If using plastic slides or staining in plastic culture dishes, do notuse xylene or xylene-based mounting media, because they dissolveplastics.7. Add one or two drops of mounting medium and cover with a coverslip.If alcohols cannot be used, mount the coverslip with glycerol or otheraqueous mounting media.

Reagents

Cells or tissue of interest on microscope slide (see Step 1.i)Eosin Y (1% aqueous solution; EM Diagnostic Systems)

Ethanol (95%, 100%)

Methanol or Flex alcohols (Richard-Allan Scientific) can be used insteadof ethanol (see Step 5).

Hematoxylin, Mayer's (Sigma)

Mayer's hematoxylin is the easiest to use and is compatible with mostcolorimetric substrates.Mounting medium (Canada Balsam, Sigma C1795)Use glycerol or other aqueous mounting media if alcohols cannot be used(see Step 7).

Xylene

Cryosectioning of Liver Tissues

The rabbit liver tissues (shown in FIG. 29 ) are perfused with normalsaline and fixed in 10% (v/v) formalin-neutralized solution (J.T. Baker,Inc., USA) for 24 hr. Afterward, the tissues are embedded in Tissue TekOCT Compound (#4583; Sakura Finetek Inc., USA). Embedded tissues are cutinto 10 μm thick slices and stained with Sudan IV and hematoxylin(Merck, USA). Briefly, the slices are washed with pure water for 1 minto remove the OCT compound, washed with 50% (v/v) ethanol for 30 sec,and then stained with 2% (w/v) Sudan IV for 1 hr. After further washingwith 50% (v/v) ethanol and pure water for 2 min, the slices arecounterstained with hematoxylin. Photographs shown in FIG. 30 areacquired using a microscope equipped with a 10-fold magnificationobjective and quantified on an Alpha Imager 2200 documentation system(Alpha Innotech, USA). The manifestation of fatty liver progression ispresented as the percentage of the area of oil droplets to the totalliver tissues (cells).

Score of Steatosis Score of Score of Slide No. Grade LocationInflammation Fibrosis ND1 0 0 0 0 ND2 0 0 0 0 ND3 0 0 0 0 HF1 3 3 2 2-3HF2 2 1-2 1 2-3 HF3 3 2-3 1 3-4 L1 2 1 2 1 L2 2 1 1 0 L3 0 0 0 0ARH003-1 3 2 1 0 ARH003-2 3 2 1 0 ARH003-3 2 2 1 0 ARH004-1 3 2 1 1ARH004-2 3 2 0 0 ARH004-3 3 2 1 0 AR101-DS2-1 3 2 1 0 AR101-DS2-2 3 1 20 AR101-DS2-3 1 0 1 0 Score of Steatosis Grade 0′: low- to medium-powerevaluation of parenchymal involvement <5% 1′: 5-33% 2′: 33-66% 3′: >66%Location 0′: zone 3, centrilobular 1′: zone 2, mid-zonal 2′: zone 3,periortal 3′: panacinar Score of Fibrosis 0′: none 1′: mildperisinusoidal or periportal 2′: perisinusoidal and portal/periportal3′: bridging fibrosis 4′: cirrhosis Score of Inflammation 0′: no foci1′: mild, 2 foci per 200 field 2′: moderate, 2-4 foci per 200 field 3′:severe, 4 foci per 200 field

Example 19. The Protective Effect of Antrodia camphorata Extract andCompounds on Bleomycin-Induced Pulmonary Fibrosis in Mice

Animals and Treatments

Specific pathogen-free ICR mice (males) (body weights 18-22 g) werepurchased from the BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan). Theanimals were housed in Plexiglas cages at a constant temperature of22±1° C. and a relative humidity of 55±5% on a 12 h dark-light cycle forat least 2 weeks before the experiment. Animals were provided food andwater ad libitum. All experimental procedures were performed accordingto the guidelines of the Institutional Animal Ethics Committee, and theprotocol was approved by the Committee for the Purpose of Control andSupervision of Experiments on Animals.

BLM-Induced PF in Mice

Mice were divided into fifth groups with 5 animals per group accordingto body weight: control group, BLM group, BLM+DEX group (7.5 mg/kg),BLM+ACH dosage group (50 mg/kg), and BLM+ACM dosage group (25 mg/kg),BLM+ACH dosage group (50 mg/kg), and BLM+ACM dosage group (25 mg/kg)BLM+AH dosage group (50 mg/kg), and BLM+AM dosage group (25 mg/kg)BLM+BH dosage group (50 mg/kg), and BLM+BM dosage group (25 mg/kg)BLM+CH dosage group (50 mg/kg), and BLM+CM dosage group (25 mg/kg)BLM+DH dosage group (50 mg/kg), and BLM+DM dosage group (25 mg/kg)BLM+EH dosage group (50 mg/kg), and BLM+EM dosage group (25 mg/kg) BLM.Pulmonary fibrosis (PF) was established in mice via a singleintratracheal administration of BLM at 7.5 mg/kg mg/kg body weight.Different doses of samples were intragastrically administered daily for21 days after BLM injury, and DEX was used as the positive control.Control and model groups received an equal volume of vehicle (0.9% NaCl)using the same schedule and route of administration.

Mouse body weights were recorded daily. Mice were sacrificed on the 21thday using excess chloral hydrate hydrochloride anesthesia. Blood wasobtained for ELISA analyses, and whole lungs were removed and weighed.The right lungs were fixed in 10% formalin, dehydrated, and embedded inparaffin. The left lungs were used to determine hydroxyproline. Thepulmonary coefficient was calculated using the following equation: lungweight/body weight×100%

Experimental Design

Male C57BL/6 mice were randomly divided into the following eight groups(n=6):

1. Group I: control;2. Group II: mice received single intraperitoneal injection of BLM (7.5mg/kg BW)3. Group III: single dose (ACH, 0.5 g/kg)4. Group IV: Single dose (ACM, 1.0 g/kg)5. Group V: purified AR101-DS1 (50 mg/kg)6. Group VI: purified AR101-DS1 (25 mg/kg)7. Group VII: purified AR101-DS2 (50 mg/kg)8. Group VIII: purified AR101-DS2 (25 mg/kg)7. Group VII: purified AR101-DS4 (50 mg/kg)8. Group VIII: purified AR101-DS4 (25 mg/kg)7. Group VII: purified AR100-DS1 (50 mg/kg)8. Group VII: purified AR100-DS1 (25 mg/kg)7. Group VII: purified ARH013-RA1 (50 mg/kg)8. Group VIII: purified ARH013-RA1 (25 mg/kg)

Sampling of BALF

Under anaesthesia, BALF was performed four times through a trachealcannula with 0.7 mL of saline. In each mouse examined, ˜2.5 mL (90%) ofBAL fluid (BALF) was recovered. The supernatants of BALF were stored at−80° C. until used.

Lung Histopathology

The anterior portion of the right lung from each mouse was fixed in 10%formaldehyde phosphate buffer, embedded in paraffin, cut into 5 μmsections, and then treated with an hematoxylin and eosin (H&E) stain forhistological examination under the light microscopy (Nikon, ECLIPSE,TS100, Tokyo, Japan). Images were captured with a digital camera(NIS-Elements D 2.30, SP4, Build 387) at an original magnification of400×.

Assay of Hydroxyproline

The contents of hydroxyproline were analyzed in lung tissue followingthe instruction of hydroxyproline assay kit (Biosource InternationalInc., Sunnyvale, Calif., USA). The pulmonary tissues of mice were groundand homogenized with 1 ml of 6 mol/L potassium chloride solution,hydrolyzed at 95° C. for 5 hours, and the pH value was adjusted to6.0-6.8. According to the instructions, the corresponding reagents wereadded to the reaction system and mixed thoroughly and then incubated for15 minutes at 60° C. After cooling, the supernatants were collectedafter centrifuging at 3500 rpm for 10 minutes. The absorbance value ofthe supernatant from the samples was measured at 550 nm by aspectrophotometer and calculated for the contents of hydroxyproline oneach group.

TNF-α, IL-6, and IL-1β Cytokines in Serum

The serum concentration of the pro-inflammatory cytokines (i.e. tumornecrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β) in serumwere assessed with relevant enzyme-linked immunosorbent assay (ELISA)kits (Biosource International Inc., Sunnyvale, Calif., USA), based onthe manufacturer's instructions.

Myeloperoxidase (MPO) Assay

The pulmonary MPO activity was a reliable index for estimating theinfiltration of inflammatory cells in lungs. The lung tissues werehomogenized and the MPO levels were detected with the kits according tomanufacturer's instruction.

Histopathological Analyses

The right lungs were embedded in paraffin wax, fixed in 10% formalin,and processed into sections. The sections were stained with haematoxylinand eosin (H&E) or subjected to Masson's trichrome staining.

Statistical Analysis

Data obtained from animal experiments were expressed as the means andstandard errors of the means (±S.E.M.). Student's t-test were used toexamine the differences among multiple groups or between two groups.Statistical significance is expressed as *p<0.05, **p<0.01 and***p<0.001.

At the end point of the entire experiment, the animal's body weight andlung weight were recorded. Compared with control animals, the bodyweight changes of bleomycin (BLM)-administered animals weresignificantly reduced. Compared with other experimental groups, the lungindex [(lung weight/body weight)×100] showed a significant increase inbleomycin-administered animals (Table 1 and FIG. 31 ). There was asignificant decrease in the lung index of ACH, BH and DH.

TABLE 1 Lung index of A. camphorata Extract and Compounds onbleomycin-induced lung fibers. Initial body Final body PulmonaryPulmonary mass/g mass/g mass/g index Normal 34.18 ± 0.39 39.73 ± 0.380.34 ± 0.01 0.86 ± 0.02  BLM 34.28 ± 0.39 35.45 ± 0.28 0.57 ± 0.01  1.60± 0.02^(###) DEX 34.24 ± 0.5  38.50 ± 0.22 0.42 ± 0.01  1.09 ± 0.02***ACH (1.0 g/kg) 34.28 ± 0.44 39.48 ± 0.24 0.36 ± 0.01 0.91 ± 0.02** ACM(0.5 g/kg) 34.35 ± 0.45 38.42 ± 0.27 0.46 ± 0.01  1.20 ± 0.02***AH(AR101-DS1) (50 mg/kg) 34.22 ± 0.39 39.02 ± 0.37 0.46 ± 0.01 1.19 ±0.01** AM(AR101-DS1) (25 mg/kg) 34.35 ± 0.48 36.45 ± 0.28 0.52 ± 0.01 1.44 ± 0.04*** BH(AR101-DS2) (50 mg/kg) 34.44 ± 0.35  39.7 ± 0.21 0.39± 0.01 1.01 ± 0.02** BM(AR101-DS2) (25 mg/kg) 34.64 ± 0.36 37.91 ± 0.300.48 ± 0.01  1.26 ± 0.03*** CH(AR101-DS4) (50 mg/kg) 34.50 ± 0.37 37.47± 0.22 0.49 ± 0.01 1.31 ± 0.01** CM(AR101-DS4) (25 mg/kg) 34.52 ± 0.2936.44 ± 0.28 0.52 ± 0.01 1.54 ± 0.04*  DH(AR100-DS1) (50 mg/kg) 34.43 ±0.25 39.12 ± 0.30 0.39 ± 0.01 1.00 ± 0.02** DM(AR100-DS1) (25 mg/kg)34.50 ± 0.33 37.33 ± 0.25 0.52 ± 0.02  1.39 ± 0.06*** EH(ARH013-RA1) (50mg/kg) 34.28 ± 0.29 37.00 ± 0.42 0.49 ± 0.02 1.32 ± 0.06**EM(ARH013-RA1) (25 mg/kg) 34.44 ± 0.31 35.86 ± 0.25 0.55 ± 0.01 1.53 ±0.03**

Example 20. Antrodia camphorata Extract and Compounds Reduced LungDysfunction and Histopathological Changes in BLM-Induced Mice

We evaluated the histopathological lung alterations in mice forexploring the therapeutic effect of A. camphorata extract and compounds.Inflammatory infiltration and integrity of organizational structureswere observed by H&E staining (FIG. 32 ); the fibrosis degree of lungtissue was performed by Masson staining (FIG. 33 ). The control groupdemonstrated some histological findings such as a thin alveolar wall,intact alveolar structure, normal alveolar septum, and less inflammatorycells infiltration in the pulmonary mesenchyme. After 21 days of BLMadministration, alveolar edema, a significant increase in septum widthand increased inflammatory cells infiltration were observed.Administration of A. camphorata extract and compounds ameliorated theinflammatory infiltration and the damaged structure in lung tissue ascompared to that of the BLM group.

Masson staining extensive stained blue in the lung tissue and septumafter 21-day post BLM administration, suggesting a severe degree ofpulmonary fibrosis in BLM group than the normal group. After A.camphorata extract and compounds treatment, the blue area was decreased,and the fibrosis degree was alleviated. At 21 days after BLM modeling,the scores of alveolitis and fibrosis were significantly decreased afterA. camphorata extract and compounds therapy. The above results suggestedthat A. camphorata extract and compounds alleviated the degree ofinflammation and fibrosis in the lungs of mice with pulmonary fibrosis.

Example 21. Pulmonary Fibrotic Markers

Hydroxyproline content is an important index for collagen deposition inthe lung tissue. To quantify the extent of pulmonary fibrosis, thehydroxyproline content in lung tissue was measured in each group and isshown in FIG. 34 . BLM obviously increased HP content (p<0.001) comparedwith the control group. A. camphorata Extract (1.0 g/kg) and AH, BH andDH reduced lung HP significant recovery (p<0.001).

Example 22. Antrodia camphorata Extract and Compounds Alerted theBleomycin-Induced Changes in Pro-Inflammatory Cytokines

Evaluation of proinflammatory cytokine TNF-α, IL-1β, IL-6 and TGF-βlevels in serum were performed by ELISA. BLM-treated kidney injury micehad significantly increased NO, TNF-α, IL-1β, and IL-6 levels in serum,compared to the control group (FIGS. 35A-34E, respectively). treatmentwith A. camphorata Extract at doses of 1.0 g/kg and compounds (BH andDH) significantly improved necrosis and inflammatory infiltrating cellsin the lung tissue treatment improved TNF-α, IL-1β, IL-6 and TGF-βproduction after BLM challenge (p<0.001).

Example 23. Effects of A. camphorata Extract and Compounds on Lung MPOActivity

As depicted in FIG. 36 , there was a remarkable increase of the MPOlevel in response to BLM challenge compared with that in control group(p<0.01). On the contrary, the administration of both A. camphorataextract, AH, BH, DH and Dex evidently suppressed the MPO activitiescompared with that in BLM group (p<0.001), which exerted stronger effectthan that in A. camphorata extract and compounds group (p<0.05) (FIG. 36).

1. A method of preventing or treating a fibrotic condition, comprisingadministering an effective amount of composition to a subject in needthereof, wherein the composition comprises 4-fused-rings triterpenesextracted from Antrodia camphorate.
 2. The method of claim 1, whereinthe 4-fused-rings triterpenes are obtained from an ethanol extract ofAntrodia camphorate.
 3. The method of claim 1, wherein the 4-fused-ringstriterpenes are obtained from an ethyl acetate extract of Antrodiacamphorate.
 4. The method of claim 1, wherein the 4-fused-ringstriterpenes are obtained from a methanol extract of Antrodia camphorate.5. The method of claim 1, wherein the 4-fused-rings triterpenes areobtained from an organic eluent by introducing a methanol extract ofAntrodia camphorate into a normal phase chromatography column, andeluting the column with hexane/ethyl acetate/methanol.
 6. The method ofclaim 5, wherein the organic eluent comprises at least one compoundselected from the group consisting following formulae and combinationsthereof:


7. A method of preventing or treating a fibrotic condition, comprisingadministering to a subject in need thereof an effective amount of acompound selected from the group consisting following formulae andcombinations thereof:


8. The method of claim 1, wherein the fibrotic condition includeshepatic fibrosis, renal fibrosis, vascular fibrosis, pulmonary fibrosis,benign prostatic hyperplasia.
 9. The method of claim 1, wherein thecomposition further alleviates renal dysfunction and renal injury. 10.The method of claim 1, wherein the composition further alleviatesnon-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease(NAFLD), and inflammation, vacuolation and necrosis in liver.
 11. Themethod of claim 7, wherein the fibrotic condition includes hepaticfibrosis, renal fibrosis, vascular fibrosis, pulmonary fibrosis, benignprostatic hyperplasia.
 12. The method of claim 7, wherein the compoundfurther alleviates renal dysfunction and renal injury.
 13. The method ofclaim 7, wherein the compound further alleviates non-alcoholicsteatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), andinflammation, vacuolation and necrosis in liver.